The overall goal of this proposal is to delineate the ex vivo serum-free culture conditions to expand CD34+ cells derived from cryopreserved umbilical cord blood cells to levels that will support its use in the adult transplant setting. The disadvantage of umbilical cord blood for adult transplants has been attributed to having too few long-term engrafting cells/hematopoietic stem cells in the cord blood. Studies presented herein suggest that this problem can be circumvented based on 1) the development of a clinical grade serum-free medium designated especially for the ex vivo expansion of CD 34+ cells derived from cord blood, 2) the successful expansion of CD 34+ cells from cryopreserved umbilical cord blood to levels adequate to support adult transplantation using the clinical grade serum-free medium with the early acting cytokines, Flt-3 ligand, stem cell factor, thrombopoietin, and Interleukin 6, and 3) the ability of these ex vivo cultured CD 34+ cells to engraft long-term in the fetal sheep model for human hematopoiesis. The Phase I milestone will be to repeat and extended these initial studies and assess the long-term engrafting ability of these cultured CD 34+ cells in increased numbers of animals and evaluate longer culture periods. These critical results will form the basis of the Phase II proposal to determine if 1) the long-term engrafting cells are being expanded or maintained under these ex vivo serum-free culture conditions, 2) define the optimal culture period yielding high quality long-term engrafting cells, and 3) evaluate other cytokines in combination with the above cytokine platform. The ability to expand the long-term engrafting cells would be invaluable in the adult transplant setting. The efforts from this proposal will yield clinically relevant data on the optimal conditions necessary for use of cryopreserved cord blood for adult transplantation. . The products derived form this proposal will support the ex vivo culture of CD34+ cells derived from cryopreserved umbilical cord blood and the maintenance of these cells ability to engraft long-term. Since cord blood has few long-term engrafting cells /hematopoietic stem cells it can only be used for infant transplantation, not adult transplantation. The ability to develop a serum-free clinical grade culture system that will support the expansion of the long-term engrafting cells will facilitate the clinical use of these cells in adult transplants.